DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first alpha-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the alpha/beta-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in alpha/beta-hydrolases.
The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (betanuc approximately 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.
Title: Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad Ruzzini AC, Ghosh S, Horsman GP, Foster LJ, Bolin JT, Eltis LD Ref: J Am Chem Soc, 134:4615, 2012 : PubMed
Meta-cleavage product (MCP) hydrolases are members of the alpha/beta-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6A resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.
Title: The molecular basis for inhibition of BphD, a C-C bond hydrolase involved in polychlorinated biphenyls degradation: large 3-substituents prevent tautomerization Bhowmik S, Horsman GP, Bolin JT, Eltis LD Ref: Journal of Biological Chemistry, 282:36377, 2007 : PubMed
The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using WT BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A:3-Cl HOPDA and S112A:3,10-diF HOPDA complexes revealed a nonproductive binding mode in which the plane defined by the C atoms of HOPDA's dienoate moiety is nearly orthogonal to that of the proposed keto tautomer observed in the S112A:HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A:3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.
Title: The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad Horsman GP, Bhowmik S, Seah SY, Kumar P, Bolin JT, Eltis LD Ref: Journal of Biological Chemistry, 282:19894, 2007 : PubMed
BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.
Title: Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway Horsman GP, Ke J, Dai S, Seah SY, Bolin JT, Eltis LD Ref: Biochemistry, 45:11071, 2006 : PubMed
Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.
Title: Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls Seah SY, Terracina G, Bolin JT, Riebel P, Snieckus V, Eltis LD Ref: Journal of Biological Chemistry, 273:22943, 1998 : PubMed
2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The kcat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.
